Particles for detecting intracellular targets

ABSTRACT

Methods describing the use of nanoparticles modified with binding moieties are provided.

GRANT FUNDING DISCLOSURE

This invention was made with government support under grant numbersIU54-CA 119341, awarded by the Cancer Center for NanotechnologyExcellence (NCl/CCNE) and 5DPIOD000285, awarded from a NIH Director'sPioneer Award, and grant number EEC-0647560, awarded by the NSEC. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods of detecting the intracellularconcentration of a target molecule using a nanoparticle wherein thenanoparticle comprises a binding moiety that can specifically associatewith the target molecule, and wherein said association results in achange in a detectable marker that can be measured after associationwith the target molecule.

BACKGROUND OF THE INVENTION

Labeled oligonucleotides are widely used probes for detecting specificmacromolecule targets such as nucleic acids and proteins. Their abilityto bind targets with high specificity has rendered them useful in invitro assays such as polymerase chain reaction (PCR) protocols. However,delivering these types of target specific probes into living cellsremains a major challenge as cells are both naturally resistant tonucleic acid uptake and contain a variety of pathways to remove theseforeign genetic materials. Thus, methods that deliver these materialsinto cells, in a manner in which they retain both their specific bindingproperties and fluorescent signaling ability are of great interest.

The discovery and subsequent development of theoligonucleotide-nanoparticle conjugate have lead to a variety of newopportunities in molecular diagnostics (Elghanian et al., 1997, Science277: 1078-1081; Nam et al., 2003, Science 308: 1884-1886) and materialsdesign (Mirkin et al., 1996, Nature 382: 607-609; Alivisatos et al.,1996, Nature 382: 609-611; Demers et al., 2003, Angew. Chem. Int. Ed.40: 3071-3073). Recently, it has been demonstrated thatoligonucleotide-functionalized nanoparticles enter cells and act asantisense agents to control gene expression (Rosi et al., 2006, Science312: 1027-1030). These “antisense particles” are not simply deliveryvehicles (Sandhu et al., 2002, Bioconjugate Chem. 13: 3-6; Tkachenko etal., 2003, J. Am. Chem. Soc. 125: 4700-4701), but rather single entityregulation and transfection agents that undergo cellularinternalization, resist enzymatic degradation, and bind intracellulartargets with affinity constants that are as much as two orders ofmagnitude greater than free oligonucleotides (Lytton-Jean and Mirkin,2005, J. Am. Chem. Soc. 127: 12754-12755). Moreover, they can be easilymodified with potent, i.e., highly stable, designer materials such aslocked nucleic acids (Seferos et al., 2007, Chem Bio Chem 8: 1230-1232)and are nontoxic under conditions required for gene regulation. Indeed,it has been shown that, unlike oligonucleotides free in solution,oligonucleotide-modified gold nanoparticles are readily taken up bycells in high numbers. This property has lead to the discovery thatoligonucleotide-modified gold nanoparticles can be used as agents forintracellular gene control, where they provide rapid intracellulardelivery of DNA, and further increase the efficacy of theoligonucleotides in the cells based on cooperative properties. Theseoligonucleotide functionalized nanoparticles have been shown to enter avariety of cell types, and can be used to introduce high localconcentrations of oligonucleotides.

It has also previously been shown that gold nanoparticles that aredensely functionalized with DNA bind complementary DNA in a highlycooperative manner, resulting in a binding strength that is twoorders-of-magnitude greater than that determined for analogous DNAstrands that are not attached to a gold nanoparticle. This property hasrendered nanoparticles particularly useful for DNA and proteindiagnostic assays in addition to those uses described above.

One class of oligonucleotides of interest are those that can detect aspecific target with a recognition sequence. These types of structures,if introduced into living cells, are especially of interest for medicaldiagnosis, drug discovery, and developmental and molecular biologyapplication. However, current delivery/transfection strategies lack theattributes required for their use such as 1) low toxicity, 2) highcellular uptake, and 3) provide resistance to enzymes that lead to falsepositive signals.

Probes to visualize and detect intracellular RNA including those usedfor in situ staining (Femino et al., Science 280: 585-590, 1998;Kloosterman et al., Nat. Methods 3: 27-29, 2006), molecular beacons(Tyagi et al., 1996, Nat. Biotechnol. 14: 303-308; Sokol et al., 1998,Proc. Natl. Acad. Sci. USA 95: 11538-11543; Peng et al., 2005, CancerRes. 65: 1909-1917; Perlette et al., 2001, Anal. Chem. 73: 5544-5550;Nitin et al., 2004, Nucleic Acids Res. 32: e58), and FRET-pairs(Santangelo et al., 2004, Nucleic Acids Res. 32: e57; Bratu et al.,2003, Proc. Natl. Acad. Sci. USA 100: 3308-13313) each of which areimportant biological tools to measure and quantify activity in livingsystems in response to external stimuli (Santangelo et al., 2006, Annalsof Biomedical Engineering 34: 39-50). However, the delivery ofoligonucleotide-based reporters into cellular media and cells has provento be a major challenge for intracellular detection. The cellularinternalization of oligonucleotide-based probes typically requirestransfection agents such as lipids (Zabner et al., 1995, J. Bio. Chem.270: 18997-19007) or dendrimers (Kukowska-Latallo et al., 1996, Proc.Natl. Acad. Sci. USA 93: 4897-4902) which can be toxic or alter cellularprocesses. Furthermore, oligonucleotides are prone to degradation withincells (Opalinska and Gewirtz, 2002, Nat. Rev. Drug Disc. 1: 503-514),and in the case of fluorophore-labeled probes, this can lead to a highbackground signal that is indistinguishable from a true recognitionevent (Li et al., 2004, Nucleic Acids Res. 28: e52; Rizzo et al., 2002,Molecular and Cellular Probes 16: 277-283).

Accordingly, while nanoparticle have been designed that can recognizetargets with a high degree of specificity, it is difficult to detect apositive effect arising from the specific interaction, particularly withthe sensitivity to detect such an interaction at the single cell level.

Thus there exists a need in the art to develop materials which arecapable of entering a cell to associate with a specific target andmethods to detect and quantitate the resulting intracellularinteraction.

SUMMARY OF THE INVENTION

Provided here are methods of determining the intracellular concentrationof a target molecule comprising the step of contacting the targetmolecule with a nanoparticle under conditions that allow association ofthe target molecule with the nanoparticle, the nanoparticle comprising abinding moiety specific for said target molecule, the binding moietylabeled with a marker, wherein the association of the target moleculeand the nanoparticle results in detectable change in the marker, andwherein the change in the detectable marker is proportional to theintracellular concentration of said target molecule.

In one embodiment of the methods, the binding moiety is a polynucleotideand in another aspect, the binding moiety is a polypeptide. In theembodiment wherein the binding moiety is a polynucleotide, alternativeaspects include those in which the binding moiety is a DNA molecule oran RNA molecule. In other embodiments of the methods, the targetmolecule is a polynucleotide or a polypeptide. In the embodiment whereinthe target molecule is a polynucleotide, alternative aspects includethose in which the binding moiety which is a DNA molecule or an RNAmolecule.

In one embodiment, methods are provided wherein the binding moiety is apolynucleotide covalently attached to the nanoparticle and the marker isa label attached to a polynucleotide hybridized to the binding moietypolynucleotide, wherein association of the binding moiety polynucleotidewith the target molecule releases the hybridized polynucleotide and themarker is detectable after release. In one aspect, the marker isattached to the hybridized polynucleotide and the marker is quenchedwhen the hybridized polynucleotide with the marker is hybridized to thebinding moiety.

In another embodiment, methods are provided wherein the binding moietyis a polypeptide covalently attached to the nanoparticle and the markeris a label attached to an agent associated with the binding moietypolypeptide, wherein association of the binding moiety polypeptide withthe target molecule displaces the associated agent and the marker isdetectable after release. In one aspect, the marker is attached to theagent and the marker is quenched when the agent is associated with thebinding moiety polypeptide.

In another embodiment, methods are provided wherein the binding moietyis labeled with a marker and the marker is detectable only when bindingmoiety is associated with the target molecule. In the embodiment whereinthe binding moiety is a polynucleotide, alternative aspects includethose in which the binding moiety is a DNA molecule or an RNA molecule.In other embodiments of the methods, the target molecule is apolynucleotide or a polypeptide. In the embodiment wherein the targetmolecule is a polynucleotide, alternative aspects include those in whichthe binding moiety which is a DNA molecule or an RNA molecule.

In one aspect, the binding moiety is a polynucleotide and the marker isattached to the polynucleotide binding moiety such that the marker isquenched when the polynucleotide binding moiety is not associated withthe target molecule. Accordingly, the marker attached to thepolynucleotide binding moiety is detectable only when the polynucleotidebinding moiety is associated with the target molecule.

In another aspect, the binding moiety is a polypeptide and the marker isattached to polypeptide binding moiety such that the marker is quenchedwhen the polypeptide binding moiety is not associated with the targetmolecule. Accordingly, the marker attached to the polypeptide bindingmoiety is detectable only when the polypeptide biding moiety isassociated with the target molecule.

Also provided are methods wherein said nanoparticle comprises amultiplicity of binding moieties. In one aspect, methods are providedwherein the multiplicity of binding moieties specifically associate withone target molecule. In another aspect, the multiplicity bindingmoieties specifically associate with more than one target molecule.

Further aspects of the invention will become apparent from the detaileddescription provided below. However, it should be understood that thefollowing detailed description and examples, while indicating preferredembodiments of the invention, are given by way of illustration onlysince various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description.

DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a scheme for the gold nanoparticles modified with afluorophore containing oligonucleotide that is capable of detecting anintracellular target.

FIG. 2 shows nano-flares for mRNA detection and quantification.

FIG. 3 describes quantification of survivin knockdown using nano-flares.(a) Flow cytometry data collected on siRNA treated SKBR3 cells. (b) Plotof mean fluorescence (black circles) and survivin expression (greybar-graph) as a function of siRNA concentration.

DETAILED DESCRIPTION OF THE INVENTION

Methods provided herein exploit the physical properties and applicationsof nanoparticles modified to include one or more binding moieties thatspecifically recognize and associate with one or more specific targetmoieties. It is shown herein that intracellular concentrations of targetmolecules can be determined using nanoparticles that comprise a bindingmoiety that is specific for the target molecule. In the presentinvention the binding moiety is labeled with a marker wherein theassociation of the target molecule and the nanoparticle results indetectable change in the marker, and wherein the change in thedetectable marker is proportional to the intracellular concentration ofsaid target molecule. It will be appreciated that the intracellulartarget includes those which are naturally occurring in the target cell,those which are naturally occurring targets that have been introducedinto the cell (but are not ordinarily found in that cell type) orsynthetic targets which do not occur in nature but have been introducedinto the target cell.

In another aspect of the present invention, the intracellularlocalization of a desired target may also be determined using themethods outlined herein.

As used herein, the term “binding moiety” is understood to encompass apolynucleotide or a polypeptide, or any other fragment or segment of anyof the preceding molecules that can associate with a target of interest.This term includes, but is not limited to, small molecules of interest.As is understood in the art, the term “small molecule” includes organicand inorganic compounds which are either naturally-occurring compounds,modifications of naturally-occurring compounds, or synthetic compounds.

The methods provided are particularly amenable to use of bindingmoieties which recognize and associate with intracellular targetmolecules, wherein the binding moieties are polynucleotides and/orpolypeptides, and the target molecules are polynucleotides and/orpolypeptides. In a simple aspect, a polynucleotide binding moietyspecifically associates with a polynucleotide target molecule or apolypeptide binding moiety specifically associates with a polypeptidetarget molecule. However, methods are also contemplated wherein apolynucleotide binding moiety specifically associates with a polypeptidetarget molecule or a polypeptide binding moiety specifically associateswith a polynucleotide target molecule.

As used herein, the term “specifically recognizes” or “specificallyassociates” means that the binding moiety can identify and/or interactwith one target molecule with a higher affinity and/or avidity comparedto all other target molecules.

The methods provided function under the principle that the bindingmoiety is directly or indirectly labeled with a marker, and associationof the binding moiety with the target molecule results in the markerbecoming detectable, or more detectable. Accordingly, when the bindingmoiety is not associated with the target molecule, the marker isrelatively undetectable, or quenched. While it is understood in the artthat the term “quench” or “quenching” is often associated withfluorescent markers, it is contemplated herein that the signal of anymarker that is quenched when it is relatively undetectable. Thus, it isto be understood that methods exemplified throughout this descriptionthat employ fluorescent markers are provided only as single embodimentsof the methods contemplated, and that any marker which can be quenchedcan be substituted for the exemplary fluorescent marker.

In one aspect, the marker is a label attached directly to the bindingmoiety, and in another aspect, the marker is a label attached to anagent associated with the binding moiety, this agent having a lowerbinding affinity or binding avidity for the binding moiety such thatassociation of the target molecule with the binding moiety causes theagent to be displaced from its association with the binding moiety.

When the marker is attached directly to the binding moiety, the markeris positioned such that it is relatively undetectable or quenched whenthe binding moiety is not associated with a target molecule. Forexample, with a polynucleotide binding moiety that is not associatedwith a target molecule, the marker can be positioned in proximity to thenanoparticle itself through either secondary structure which formswithin the polynucleotide binding moiety, or the marker can be freelywavering in the aqueous environment such that at any given time themarker can be in proximity to the nanoparticle and its signal quenchedor freely wavering (though still tethered to the nanoparticle) in theaqueous environment at a distance from the nanoparticle that the signalis not quenched. In this embodiment wherein no secondary structure holdsthe marker in a quenched position in proximity to the nanoparticle whenthe polynucleotide binding moiety is not in association with a targetmolecule, a level of background signal will necessarily be detected, andassociation of the polynucleotide binding moiety with a target moleculewill strengthen the signal over background as a result of the fact thatmore marker with be displaced from sufficient proximity to thenanoparticle to impart a quenching effect.

Similarly when the binding moiety is a polypeptide, the marker on thebinding moiety may be positioned such that a conformation change thatoccurs when the polypeptide binding moiety is in association with atarget molecule results in the marker moving sufficiently away from thenanoparticle that its signal is relatively unquenched.

In aspects of the methods wherein the marker is indirectly associatedwith the binding moiety, association of the binding moiety with a targetmolecule cause a physical release of the marker such that thenanoparticle is no longer able to exert a quenching effect on themarker. For example, with a polynucleotide binding moiety, the markermay be labeled on a second polynucleotide which can hybridize to thepolynucleotide binding moiety in a position such that the marker is insufficient proximity to the nanoparticle that the nanoparticle exertsits quenching effect. When the polynucleotide binding moleculerecognizes and associates with a target molecule, the hybridized andlabeled polynucleotide is displaced, and the quenching effect of thenanoparticle is abated.

Thus, in one aspect for example, methods are provided wherein goldnanopa modified to include a polynucleotide binding moiety which in turnis hybridized to complementary polynucleotide labeled with a fluorophoremarker can be used as both transfection agents and cellular“nano-flares” for visualizing and quantifying RNA in living cells.Nano-flares take advantage of the highly efficient fluorescencequenching properties of gold (Dubertret et al., 2001, Nat. Biotechnol.19: 365-370), cellular uptake of oligonucleotide nanoparticle conjugateswithout the use of transfection agents, and the enzymatic stability ofsuch conjugates (Rosi et al., 2006, Science 312: 1027-1030), thusovercoming many of the challenges to creating sensitive and effectiveintracellular probes. Specifically, nano-flares exhibit high signaling,have low background fluorescence, and are sensitive to changes in thenumber of RNA transcripts present in cells. Thus, the nano-flaresdescribed herein are oligonucleotide functionalized nanoparticleconjugates designed to provide an intracellular fluorescence signal thatdirectly or indirectly correlates with the relative amount of a specificintracellular RNA. RNA contemplated for detection in the disclosedmethods include, but are not limited to, mRNA and hnRNA.

A similar mechanism operates for a polypeptide binding moiety, whereinan agent labeled with a marker and the agent is able to associate withthe polypeptide binding moiety in such a way that its association bringsthe agent and marker sufficiently close to the nanoparticle that themarker is relatively quenched. When the polypeptide binding moietyassociates with a specific target molecule, the agent is released ordisplaced as with the nano-flare described above and the quenchingeffect of the nanoparticle is relieved.

Regardless of the specific nature of the binding moiety, by utilizingnanoparticles densely functionalized with fluorophore-labeledoligonucleotides or polypeptides, several difficulties commonlyassociated with intracellular molecule detection are alleviated. Thesebinding moieties do not require microinjection or auxiliary transfectionreagents to enter cells, are highly resistant towards enzymaticdegradation and are non-toxic under the conditions studied.

Polynucleotides

As used herein, the term in “polynucleotide,” either functionalized on ananoparticle or as a target molecule, is used interchangeably with theterm oligonucleotide.

The term “nucleotide” or its plural as used herein is interchangeablewith modified forms as discussed herein and otherwise known in the art.In certain instances, the art uses the term “nucleobase” which embracesnaturally-occurring nucleotides as well as modifications of nucleotidesthat can be polymerized.

Methods of making polynucleotides of a predetermined sequence arewell-known in the art. See, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotidesand Analogues, 1st Ed. (Oxford University Press, New York, 1991).Solid-phase synthesis methods are preferred for botholigoribonucleotides and oligodeoxyribonucleotides (the well-knownmethods of synthesizing DNA are also useful for synthesizing RNA).Oligoribonucleotides and oligodeoxyribonucleotides can also be preparedenzymatically.

In various aspects, methods provided include use of polynucleotideswhich are DNA oligonucleotides, RNA oligonucleotides, or combinations ofthe two types. Modified forms of oligonucleotides are also contemplatedwhich include those having at least one modified internucleotidelinkage. Modified polynucleotides or oligonucleotides are described indetail herein below.

Modified Oligonucleotides

Specific examples of oligonucleotides include those containing modifiedbackbones or non-natural internucleoside linkages. Oligonucleotideshaving modified backbones include those that retain a phosphorus atom inthe backbone and those that do not have a phosphorus atom in thebackbone. Modified oligonucleotides that do not have a phosphorus atomin their internucleoside backbone are considered to be within themeaning of “oligonucleotide.”

Modified oligonucleotide backbones containing a phosphorus atom include,for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Also contemplated are oligonucleotides having inverte polaritycomprising a single 3′ to 3′ linkage at the 3′-most internucleotidelinkage, i.e. a single inverted nucleoside residue which may be abasic(the nucleotide is missing or has a hydroxyl group in place thereof).Salts, mixed salts and free acid forms are also contemplated.Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, U.S. Pat. Nos. 3,687,808;4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599;5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, thedisclosures of which are incorporated by reference herein.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages; siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; riboacetyl backbones;alkene containing backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts. See,for example, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134;5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704;5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and5,677,439, the disclosures of which are incorporated herein by referencein their entireties.

In still other embodiments, oligonucleotide mimetics wherein both one ormore sugar and/or one or more internucleotide linkage of the nucleotideunits are replaced with “non-naturally occurring” groups. In one aspect,this embodiment contemplates a peptide nucleic acid (PNA). In PNAcompounds, the sugar-backbone of an oligonucleotide is replaced with anamide containing backbone. See, for example U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, and Nielsen et al., 1991, Science, 254:1497-1500, the disclosures of which are herein incorporated byreference.

In still other embodiments, oligonucleotides are provided withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and including —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂—,—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—described in U.S. Pat. Nos. 5,489,677, and 5,602,240. Also contemplatedare oligonucleotides with morpholino backbone structures described inU.S. Pat. No. 5,034,506.

In various forms, the linkage between two successive monomers in theoligo consists of 2 to 4, desirably 3, groups/atoms selected from —CH₂—,—O—, —S—, —NR^(H)—, >C═O, >C═NR^(H), >C═S, —Si(R″)₂—, —SO—, —S(O)₂—,—P(O)₂—, —PO(BH₂)—, —P(O,S)—, —P(S)₂—, —PO(R″)—, —PO(OCH₃)—, and—PO(NHR^(H))—, where RH is selected from hydrogen and C₁₋₄-alkyl, and R″is selected from C₁₋₆-alkyl and phenyl. Illustrative examples of suchlinkages are —CH₂—CH₂—CH₂—, —CH₂—CO—CH₂—, —CH₂—CHOH—CH₂—, —O—CH₂—O—,—O—CH₂—CH₂—, —O—CH₂—CH=(including R⁵ when used as a linkage to asucceeding monomer), —CH₂—CH₂—O—, —NR^(H)—CH₂—CH₂—, —CH₂—CH₂—NR^(H)—,—CH₂—NR^(H)—CH₂—, —O—CH₂—CH₂—NR^(H)—, —NR^(H)—CO—O—, —NR^(H)—CO—NR^(H)—,—NR^(H)—CS—NR^(H)—, —NR^(H)—C(═NR^(H))—NR^(H)—,—NR^(H)—CO—CH₂—NR^(H)—O—CO—O—, —O—CO—CH₂—O, —O—CH₂—CO—O—,—CH₂—CO—NR^(H)—, —O—CO—NR^(H)—, —NR^(H)CO—CH₂—, —O—CH₂—CO—NR^(H)—,—O—CH₂—CH₂—NR^(H)—, —CH═N—O—, —CH₂—NR^(H)—O—, —CH₂—O—N=(including R⁵when used as a linkage to a succeeding monomer), CH₂—O—NR^(H)—,—CO—NR^(H)—CH₂—, —CH₂—NR^(H)—O—, —CH₂—NRH—CO—, —O—NR^(H)—CH₂—,—O—NR^(H), —O—CH₂—S—, —S—CH₂—O—, —CH₂—CH₂S—, —O—CH₂—CH₂—S—,—S—CH₂—CH=(including R⁵ when used as a linkage to a succeeding monomer),—S—CH₂—CH₂—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—S—CH₂—, —CH₂—SO—CH₂—,—CH₂—SO₂—CH₂—, —O—SO—O—, —O—S(O)₂—O—, —O—S(O)₂—CH₂—, —O—S(O)₂—NR^(H)—,—NR^(H)—S(O)₂—CH₂; —O—S(O)₂—CH₂—, —O—P(O)₂—O—, —P(O,S)—O—, —O—P(S)₂—,—S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—,—O—P(S)₂—S—, —S—P(O)₂—S—, —S—P(O,S)—S—, —S—P(S)₂—S—, —O—PO(R″)—O—,—O—PO(OCH₃)—O—, —O—PO(OCH₂CH₃)—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(N))—O—, —O—P(O)₂—NR^(H)H—H—, —NR^(H)—P(O)₂—O—,—O—P(O,NR^(H))—O—, —CH₂—P(O)₂—O—, —O—P(O)₂—CH₂—, and —O—Si(R″)₂—O—;among which —CH₂—CO—NR^(H)—, —CH₂—NR^(H)—O—, —S—CH₂—O—,—O—P(O)₂—O—O—P(—O,S)—O—, —O—P(S)₂—O—, —NR^(H)P(O)₂—O—,—O—P(O,NR^(H))—O—, —O—PO(R″)—O—, —O—PO(CH₃)—O—, and —O—PO(NHR^(N))—O—,where RH is selected form hydrogen and C₁₋₄-alkyl, and R″ is selectedfrom C₁₋₆-alkyl and phenyl, are contemplated. Further illustrativeexamples are given in Mesmaeker et. al., 1995, Current Opinion inStructural Biology, 5: 343-355 and Susan M. Freier and Karl-HeinzAltmann, 1997, Nucleic Acids Research, vol 25: pp 4429-4443.

Still other modified forms of oligonucleotides are described in detailin U.S. patent application NO. 20040219565, the disclosure of which isincorporated by reference herein in its entirety.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. In certain aspects, oligonucleotides comprise one of thefollowing at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Other embodiments includeO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other oligonucleotides comprise one of the following atthe 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃,OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH2,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.In one aspect, a modification includes T-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., 1995,Helv. Chim. Acta, 78: 486-504) i.e., an alkoxyalkoxy group. Othermodifications include 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples herein below,and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples herein below.

Still other modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl(2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. In one aspect, a2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, for example, at the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. See, for example, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; 5,792,747; and 5,700,920, the disclosures of whichare incorporated by reference in their entireties herein.

In one aspect, a modification of the sugar includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring, thereby forming a bicyclic sugar moiety. Thelinkage is in certain aspects is a methylene (—CH₂—)_(n) group bridgingthe 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

Oligonucleotides may also include base modifications or substitutions.As used herein, “unmodified” or “natural” bases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified bases include other synthetic andnatural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and3-deazaguanine and 3-deazaadenine. Further modified bases includetricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzox-azin-2(3H)-one),carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiedbases may also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further bases includethose disclosed in U.S. Pat. No. 3,687,808, those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. 1., ed. John Wiley & Sons, 1990, those disclosed byEnglisch et al., 1991, Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these bases are useful for increasingthe binding affinity and include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. and are, in certain aspects combinedwith 2′-O-methoxyethyl sugar modifications. See, U.S. Pat. No.3,687,808, U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273;5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177;5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617;5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,750,692 and 5,681,941, thedisclosures of which are incorporated herein by reference.

A “modified base” or other similar term refers to a composition whichcan pair with a natural base (e.g., adenine, guanine, cytosine, uracil,and/or thymine) and/or can pair with a non-naturally occurring base. Incertain aspects, the modified base provides a T_(m) differential of 15,12, 10, 8, 6, 4, or 2° C. or less. Exemplary modified bases aredescribed in EP 1 072 679 and WO 97/12896.

By “nucleobase” is meant the naturally occurring nucleobases adenine(A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well asnon-naturally occurring nucleobases such as xanthine, diaminopurine,8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine,N⁴,N⁴-ethanocytosin, N′,N′-ethano-2,6-diaminopurine, 5-methylcytosine(mC), 5-(C³-C⁶)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine,isoguanine, inosine and the “non-naturally occurring” nucleobasesdescribed in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freierand Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp4429-4443. The term “nucleobase” thus includes not only the known purineand pyrimidine heterocycles, but also heterocyclic analogues andtautomers thereof Further naturally and non-naturally occurringnucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan,et al.), in Chapter 15 by Sanghvi, in Antisense Research andApplication, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, inEnglisch et al., 1991, Angewandte Chemie, International Edition, 30:613-722 (see especially pages 622 and 623, and in the ConciseEncyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed.,John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design1991, 6, 585-607, each of which are hereby incorporated by reference intheir entirety). The term “nucleosidic base” or “base unit” is furtherintended to include compounds such as heterocyclic compounds that canserve like nucleobases including certain “universal bases” that are notnucleosidic bases in the most classical sense but serve as nucleosidicbases. Especially mentioned as universal bases are 3-nitropyrrole,optionally substituted indoles (e.g., 5-nitroindole), and optionallysubstituted hypoxanthine. Other desirable universal bases include,pyrrole, diazole or triazole derivatives, including those universalbases known in the art.

Polypeptides

As used herein, the term “polypeptide” refers to peptides, proteins,polymers of amino acids, hormones, viruses, and antibodies that arenaturally derived, synthetically produced, or recombinantly produced.Polypeptides also include lipoproteins and post translationally modifiedproteins, such as, for example, glycosylated proteins, as well asproteins or protein substances that have D-amino acids, modified,derivatized, or non-naturally occurring amino acids in the D- orL-configuration and/or peptomimetic units as part of their structure.

Peptides contemplated for use in the methods provided include thosederived from commercially available sources. Libraries includestructured peptide libraries comprising small, disulfide-constrainedcyclic peptide compounds that range in size from six to twelve aminoacids, wherein the number of distinct peptide structures in each librarytypically exceeds 1 billion; (ii) linear peptide libraries wherein 19amino acids (no cysteine) at each position in a 20-mer peptide areallowed to create a library of 10 billion peptides; (iii) substratephage peptide libraries wherein all 19 amino acids (no cysteine) at eachposition in a 13-mer peptide are allowed to create a library ofapproximately 100 million peptides.

Commercially available peptide libraries include those from Peptidelibraries Eurogentec s.a. (Belgium), Dyax Corp. (Cambridge, Mass.) andCambridge Peptide (Cambridge, UK).

Preparation of peptide libraries useful in practice of the method iswell known in the art, as described by Jung (ed) Combinatorial Peptideand Nonpeptide Libraries: A Handbook and in Devlin et al., 1990,Science, Vol 249, Issue 4967: 404-406, as well from use of commerciallyavailable synthesis kits from, for example, Sigma-Genosys.

Proteins contemplated for use in the methods provided include thosederived from synthesized proteins libraries as described in Matsuura, etal., 2002, Protein Science 11: 2631-2643, Ohuchi et al., 1998, NucleicAcids Res. October 1; 26(19): 4339-4346, WO/1999/011655, WO/1998/047343,U.S. Pat. No. 6,844,161 and U.S. Pat. No. 6,403,312. Commerciallyavailable kits for production of protein libraries are also know in theart and available from, for example, BioCat GmbH (Heidelberg).

Protein libraries useful in practice of the methods are alsocommercially available from, for example, Dyax Corp. (Cambridge, Mass.).

Detectable Marker/Label

A “marker” as used herein is interchangeable with “label” and regardlessof the type of interacting compound being identified, methods areprovided wherein polynucleotide or polypeptide complex formation isdetected by an observable change. In one aspect, complex formation givesrise to a color change which is observed with the naked eye orspectroscopically. When using gold nanoparticles, a red-to-blue colorchange occurs with nanoparticle aggregation which often is detected withthe naked eye. In the present invention, aggregation is contemplated tooccur as a result of separate nanoparticles, each containing bindingmoieties to a specific but different portion of the target molecule,bind the same target molecule.

In another aspect, polynucleotide or polypeptide complex formation givesrise to aggregate formation which is observed by electron microscopy orby nephelometry. Aggregation of nanoparticles in general gives rise todecreased plasmon resonance. In still another aspect, complex formationgives rise to precipitation of aggregated nanoparticles which isobserved with the naked eye or microscopically.

The observation of a color change with the naked eye is, in one aspect,made against a background of a contrasting color. For instance, whengold nanoparticles are used, the observation of a color change isfacilitated by spotting a sample of the hybridization solution on asolid white surface (such as, without limitation, silica or alumina TLCplates, filter paper, cellulose nitrate membranes, nylon membranes, or aC-18 silica TLC plate) and allowing the spot to dry. Initially, the spotretains the color of the hybridization solution, which ranges frompink/red, in the absence of hybridization, to purplish-red/purple, ifthere has been hybridization. On drying at room temperature or 80° C.(temperature is not critical), a blue spot develops if thenanoparticle-oligonucleotide conjugates had been linked by hybridizationprior to spotting. In the absence of hybridization, the spot is pink.The blue and the pink spots are stable and do not change on subsequentcooling or heating or over time providing a convenient permanent recordof the test. No other steps (such as a separation of hybridized andunhybridized nanoparticle-oligonucleotide conjugates) are necessary toobserve the color change.

An alternate method for visualizing the results from practice of themethods is to spot a sample of nanoparticle probes on a glass fiberfilter (e.g., Borosilicate Microfiber Filter, 0.7 micron pore size,grade FG75, for use with gold nanoparticles 13 nm in size), whiledrawing the liquid through the filter. Subsequent rinsing washes theexcess, non-hybridized probes through the filter, leaving behind anobservable spot comprising the aggregates generated by hybridization ofthe nanoparticle probes (retained because these aggregates are largerthan the pores of the filter). This technique allows for greatersensitivity, since an excess of nanoparticle probes can be used.

It will be understood that a marker contemplated will include any of thefluorophores described herein as well as other detectable markers knownin the art. For example, markers also include, but are not limited to,redox active probes, other nanoparticles, and quantum dots, as well asany marker which can be detected using spectroscopic means, i.e., thosemarkers detactable using microscopy and cytometry.

Methods of Labeling Oligonucleotides

Methods of labeling oligonucleotides with fluorescent molecules andmeasuring fluorescence are well known in the art. Suitable fluorescentmolecules are also well known in the art and include without limitation1,8-ANS (1-Anilinonaphthalene-8-sulfonic acid),1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS),5-(and-6)-Carboxy-2′,7′-dichlorofluorescein pH 9.0, 5-FAM pH 9.0, 5-ROX(5-Carboxy-X-rhodamine, triethylammonium salt), 5-ROX pH 7.0, 5-TAMRA,5-TAMRA pH 7.0, 5-TAMRA-MeOH, 6 JOE,6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0, 6-Carboxyhrodamine 6G pH7.0, 6-Carboxyrhodamine 6G, hydrochloride, 6-HEX, SE pH 9.0, 6-TET, SEpH 9.0, 7-Amino-4-methylcoumarin pH 7.0, 7-Hydroxy-4-methylcoumarin,7-Hydroxy-4-methylcoumarin pH 9.0, Alexa 350, Alexa 405, Alexa 430,Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 568, Alexa 594, Alexa647, Alexa 660, Alexa 680, Alexa 700, Alexa Fluor 430 antibody conjugatepH 7.2, Alexa Fluor 488 antibody conjugate pH 8.0, Alexa Fluor 488hydrazide-water, Alexa Fluor 532 antibody conjugate pH 7.2, Alexa Fluor555 antibody conjugate pH 7.2, Alexa Fluor 568 antibody conjugate pH7.2, Alexa Fluor 610 R-phycoerythrin streptavidin pH 7.2, Alexa Fluor647 antibody conjugate pH 7.2, Alexa Fluor 647 R-phycoerythrinstreptavidin pH 7.2, Alexa Fluor 660 antibody conjugate pH 7.2, AlexaFluor 680 antibody conjugate pH 7.2, Alexa Fluor 700 antibody conjugatepH 7.2, Allophycocyanin pH 7.5, AMCA conjugate, Amino Coumarin, APC(allophycocyanin), Atto 647, BCECF pH 5.5, BCECF pH 9.0, BFP (BlueFluorescent Protein), BO-PRO-1-DNA, BO-PRO-3-DNA, BOBO-1-DNA,BOBO-3-DNA, BODIPY 650/665-X, MeOH, BODIPY FL conjugate, BODIPY FL,MeOH, Bodipy R6G SE, BODIPY R6G, MeOH, BODIPY TMR-X antibody conjugatepH 7.2, Bodipy TMR-X conjugate, BODIPY TMR-X, MeOH, BODIPY TMR-X, SE,BODIPY TR-X phallacidin pH 7.0, BODIPY TR-X, MeOH, BODIPY TR-X, SE,BOPRO-1, BOPRO-3, Calcein, Calcein pH 9.0, Calcium Crimson, CalciumCrimson Ca2+, Calcium Green, Calcium Green-1 Ca2+, Calcium Orange,Calcium Orange Ca2+, Carboxynaphthofluorescein pH 10.0, Cascade Blue,Cascade Blue BSA pH 7.0, Cascade Yellow, Cascade Yellow antibodyconjugate pH 8.0, CFDA, CFP (Cyan Fluorescent Protein), CI-NERF pH 2.5,CI-NERF pH 6.0, Citrine, Coumarin, Cy 2, Cy 3, Cy 3.5, Cy 5, Cy 5.5,CyQUANT GR-DNA, Dansyl Cadaverine, Dansyl Cadaverine, MeOH, DAPI,DAPI-DNA, Dapoxyl (2-aminoethyl)sulfonamide, DDAO pH 9.0, Di-8 ANEPPS,Di-8-ANEPPS-lipid, DiI, DiO, DM-NERF pH 4.0, DM-NERF pH 7.0, DsRed,DTAF, dTomato, eCFP (Enhanced Cyan Fluorescent Protein), eGFP (EnhancedGreen Fluorescent Protein), Eosin, Eosin antibody conjugate pH 8.0,Erythrosin-5-isothiocyanate pH 9.0, Ethidium Bromide, Ethidiumhomodimer, Ethidium homodimer-1-DNA, eYFP (Enhanced Yellow FluorescentProtein), FDA, FITC, FITC antibody conjugate pH 8.0, FlAsH, Fluo-3,Fluo-3 Ca2+, Fluo-4, Fluor-Ruby, Fluorescein, Fluorescein 0.1 M NaOH,Fluorescein antibody conjugate pH 8.0, Fluorescein dextran pH 8.0,Fluorescein pH 9.0, Fluoro-Emerald, FM 1-43, FM 1-43 lipid, FM 4-64, FM4-64, 2% CHAPS, Fura Red Ca2+, Fura Red, high Ca, Fura Red, low Co,Fura-2 Ca2+, Fura-2, high Cu” Fura-2, no Cu, GFP (S65T), HcRed, Hoechst33258, Hoechst 33258-DNA, Hoechst 33342, Indo-1 Ca2+, Indo-1, Ca free,Indo-1, Ca saturated, JC-1, JC-1 pH 8.2, Lissamine rhodamine,LOLO-1-DNA, Lucifer Yellow, CH, LysoSensor Blue, LysoSensor Blue pH 5.0,LysoSensor Green, LysoSensor Green pH 5.0, LysoSensor Yellow pH 3.0,LysoSensor Yellow pH 9.0, LysoTracker Blue, LysoTracker Green,LysoTracker Red, Magnesium Green, Magnesium Green Mg2+, MagnesiumOrange, Marina Blue, mBanana, mCherry, mHoneydew, MitoTracker Green,MitoTracker Green FM, MeOH, MitoTracker Orange, MitoTracker Orange,MeOH, MitoTracker Red, MitoTracker Red, MeOH, mOrange, mPlum, mRFP,mStrawberry, mTangerine, NBD-X, NBD-X, MeOH, NeuroTrace 500/525, greenfluorescent Nissl stain-RNA, Nile Blue, EtOH, Nile Red, Nile Red-lipid,Nissl, Oregon Green 488, Oregon Green 488 antibody conjugate pH 8.0,Oregon Green 514, Oregon Green 514 antibody conjugate pH 8.0, PacificBlue, Pacific Blue antibody conjugate pH 8.0, Phycoerythrin, PicoGreendsDNA quantitation reagent, PO-PRO-1, PO-PRO-1-DNA, PO-PRO-3,PO-PRO-3-DNA, POPO-1, POPO-1-DNA, POPO-3, Propidium Iodide, PropidiumIodide-DNA, R-Phycoerythrin pH 7.5, ReAsH, Resorufin, Resorufin pH 9.0,Rhod-2, Rhod-2 Ca2+, Rhodamine, Rhodamine 110, Rhodamine 110 pH 7.0,Rhodamine 123, MeOH, Rhodamine Green, Rhodamine phalloidin pH 7.0,Rhodamine Red-X antibody conjugate pH 8.0, Rhodaminen Green pH 7.0,Rhodol Green antibody conjugate pH 8.0, Sapphire, SBFI-Na+, Sodium GreenNa+, Sulforhodamine 101, EtOH, SYBR Green I, SYPRO Ruby, SYTO 13-DNA,SYTO 45-DNA, SYTOX Blue-DNA, Tetramethylrhodamine antibody conjugate pH8.0, Tetramethylrhodamine dextran pH 7.0, Texas Red-X antibody conjugatepH 7.2, TO-PRO-1-DNA, TO-PRO-3-DNA, TOTO-1-DNA, TOTO-3-DNA, TRITC,X-Rhod-1 Ca2+, YO-PRO-1-DNA, YO-PRO-3-DNA, YOYO-1-DNA, and YOYO-3-DNA.

In yet another embodiment, two types of fluorescent-labeledoligonucleotides attached to two different particles can be used as longas the nanoparticles have the ability to quench the detectable markerbeing utilized. Suitable particles include polymeric particles (such as,without limitation, polystyrene particles, polyvinyl particles, acrylateand methacrylate particles), glass particles, latex particles, Sepharosebeads and others like particles well known in the art. Methods ofattaching oligonucleotides to such particles are well known androutinely practiced in the art. See Chrisey et al., 1996, Nucleic AcidsResearch, 24: 3031-3039 (glass) and Charreyre et al., 1997 Langmuir, 13:3103-3110, Fahy et al., 1993, Nucleic Acids Research, 21: 1819-1826,Elaissari et al., 1998, J. Colloid Interface Sci., 202: 251-260,Kolarova et al., 1996, Biotechniques, 20: 196-198 and Wolf et al., 1987,Nucleic Acids Research, 15: 2911-2926 (polymer/latex).

Other labels besides fluorescent molecules can be used, such aschemiluminescent molecules, which will give a detectable signal or achange in detectable signal upon hybridization.

Nanoparticles

As used herein, “nanoparticle” refers to small structures that are lessthan 10 μm, and preferably less than 5 μm, in any one dimension. Ingeneral, nanoparticles contemplated include any compound or substancewith a high loading capacity for an oligonucleotide as described herein.Nanoparticles useful in the practice of the invention include metal(e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe,CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g.,ferromagnetite) colloidal materials, as long as the nanoparticle has theability to quench the otherwise detectable marker. Other nanoparticlesuseful in the practice of the invention include ZnS, ZnO, TiO₂, AgI,AgBr, HgI₂, PbS, PbSe, ZnTe, CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs,and GaAs. The size of the nanoparticles is preferably from about 5 nm toabout 150 nm (mean diameter), more preferably from about 5 to about 50nm, most preferably from about 10 to about 30 nm. The size of thenanoparticle is contemplated to be from about 5 to about 10 nm, or about5 to about 20 nm, or about 5 to about 30 nm, or about 5 to about 40 nm,or about 5 to about 60 nm, or about 5 to about 70 nm, or about 5 toabout 80 nm, or about 5 to about 90 nm, or about 5 to about 100 nm, orabout 5 to about 110 nm, or about 5 to about 120 nm, or about 5 to about130 nm, or about 5 to about 140 nm, or about 10 to about 20 nm, or about10 to about 40 nm, or about 10 to about 50 nm, or about 10 to about 60nm, or about 10 to about 70 nm, or about 10 to about 80 nm, or about 10to about 90 nm, or about 10 to about 100 nm, or about 10 to about 110nm, or about 10 to about 120 nm, or about 10 to about 130 nm, or about10 to about 140 nm, or about 10 to about 150 nm. The nanoparticles mayalso be rods, prisms, or tetrahedra.

Thus, nanoparticles are contemplated for use in the methods whichcomprise a variety of inorganic materials including, but not limited to,metals, semi-conductor materials or ceramics as described in US patentapplication No 20030147966. For example, metal-based nanoparticlesinclude those described herein. Ceramic nanoparticle materials include,but are not limited to, brushite, tricalcium phosphate, alumina, silica,and zirconia. Organic materials from which nanoparticles are producedinclude carbon. Nanoparticle polymers include polystyrene, siliconerubber, polycarbonate, polyurethanes, polypropylenes,polymethylmethacrylate, polyvinyl chloride, polyesters, polyethers, andpolyethylene. Biodegradable, biopolymer (e.g. polypeptides such as BSA,polysaccharides, etc.), other biological materials (e.g. carbohydrates),and/or polymeric compounds are also contemplated for use in producingnanoparticles.

In practice, methods are provided using any suitable nanoparticle havingmolecules attached thereto that are in general suitable for use indetection assays known in the art to the extent and do not interferewith polynucleotide complex formation, i.e., hybridization to form adouble-strand or triple-strand complex. The size, shape and chemicalcomposition of the particles contribute to the properties of theresulting oligonucleotide-functionalized nanoparticle. These propertiesinclude for example, optical properties, optoelectronic properties,electrochemical properties, electronic properties, stability in varioussolutions, magnetic properties, and pore and channel size variation. Theuse of mixtures of particles having different sizes, shapes and/orchemical compositions, as well as the use of nanoparticles havinguniform sizes, shapes and chemical composition, is contemplated.Examples of suitable particles include, without limitation,nanoparticles, aggregate particles, isotropic (such as sphericalparticles) and anisotropic particles (such as non-spherical rods,tetrahedral, prisms) and core-shell particles such as the ones describedin U.S. patent application Ser. No. 10/034,451, filed Dec. 28, 2002 andInternational application no. PCT/US01/50825, filed Dec. 28, 2002, thedisclosures of which are incorporated by reference in their entirety.

Methods of making metal, semiconductor and magnetic nanoparticles arewell-known in the art. See, for example, Schmid, G. (ed.) Clusters andColloids (VCH, Weinheim, 1994); Hayat, M. A. (ed.) Colloidal Gold:Principles, Methods, and Applications (Academic Press, San Diego, 1991);Massart, R., IEEE Transactions On Magnetics, 17, 1247 (1981); Ahmadi, T.S. et al., Science, 272, 1924 (1996); Henglein, A. et al., J. Phys.Chem., 99, 14129 (1995); Curtis, A. C. et al., Angew. Chem. hit. Ed.Engl., 27, 1530 (1988). Preparation of polyalkylcyanoacrylatenanoparticles prepared is described in Fattal, et al., J. ControlledRelease (1998) 53: 137-143 and U.S. Pat. No. 4,489,055. Methods formaking nanoparticles comprising poly(D-glucaramidoamine)s are describedin Liu, et al., J. Am. Chem. Soc. (2004) 126:7422-7423. Preparation ofnanoparticles comprising polymerized methylmethacrylate (MMA) isdescribed in Tondelli, et al., Nucl. Acids Res. (1998) 26:5425-5431, andpreparation of dendrimer nanoparticles is described in, for exampleKukowska-Latallo, et al., Proc. Natl. Acad. Sci. USA (1996) 93:4897-4902(Starburst polyamidoamine dendrimers).

Suitable nanoparticles are also commercially available from, forexample, Ted Pella, Inc. (gold), Amersham Corporation (gold) andNanoprobes, Inc. (gold).

Also as described in US patent application No 20030147966, nanoparticlescomprising materials described herein are available commercially or theycan be produced from progressive nucleation in solution (e.g., bycolloid reaction), or by various physical and chemical vapor depositionprocesses, such as sputter deposition. See, e.g., HaVashi, (1987) Vac.Sci. Technol. July/August 1987, A5(4):1375-84; Hayashi, (1987) PhysicsToday, December 1987, pp. 44-60; MRS Bulletin, January 1990, pgs. 16-47.

As further described in US patent application No 20030147966,nanoparticles contemplated are produced using HAuCl₄ and acitrate-reducing agent, using methods known in the art. See, e.g.,Marinakos et al., (1999) Adv. Mater. 11: 34-37; Marinakos et al., (1998)Chem. Mater. 10: 1214-19; Enustun & Turkevich, (1963) J. Am. Chem. Soc.85: 3317. Tin oxide nanoparticles having a dispersed aggregate particlesize of about 140 nm are available commercially from VacuumMetallurgical Co., Ltd. of Chiba, Japan. Other commercially availablenanoparticles of various compositions and size ranges are available, forexample, from Vector Laboratories, Inc. of Burlingame, Calif.

Nanoparticle Functionalized with Structure-Switching RecognitionSequence

In other embodiments, the detectable change is created by labeling theoligonucleotides with molecules (e.g., and without limitation,fluorescent molecules and dyes) that produce detectable changes uponhybridization of the oligonucleotides on the nanoparticles. In oneaspect, for example, oligonucleotides or polypeptides functionalized onnanoparticles have a marker attached to the terminus distal to thenanoparticle attachment terminus, and in the absence of association witha target, the distal terminus with the marker is positioned in proximityto the nanoparticle close enough to quench fluorescence of the marker.In one aspect, metal and semiconductor nanoparticles are knownfluorescence quenchers, with the magnitude of the quenching effectdepending on the distance between the nanoparticles and the fluorescentmolecule. Thus, in the single-strand state, the oligonucleotidesattached to the nanoparticles interact with the nanoparticles through,e.g., a hairpin structure formed by the oligonucleotide throughsecondary structure folding, which brings the fluorescent molecule inproximity to the nanoparticle, so that significant quenching isobserved. Similarly, in the unbound state, polypeptides attached to thenanoparticles would assume a conformation that would bring the markerinto proximity with the nanoparticle and the marker would be quenched.Upon polynucleotide or polypeptide complex formation due to targetmolecule binding via the recognition sequence, the fluorescent moleculewill become spaced away from the nanoparticles, diminishing quenching ofthe fluorescence (FIG. 1). Useful lengths of the oligonucleotides can bedetermined empirically. Thus, in various aspects, metallic andsemiconductor nanoparticles having fluorescent-labeled oligonucleotidesor polypeptides attached thereto are used in any of the assay formatsdescribed herein.

Attaching Oligonucleotides to Nanoparticles

Nanoparticles for use in the methods provided are functionalized with anoligonucleotide, or modified form thereof, which is from about 5 toabout 100 nucleotides in length. Methods are also contemplated whereinthe oligonucleotide is about 5 to about 90 nucleotides in length, about5 to about 80 nucleotides in length, about 5 to about 70 nucleotides inlength, about 5 to about 60 nucleotides in length, about 5 to about 50nucleotides in length about 5 to about 45 nucleotides in length, about 5to about 40 nucleotides in length, about 5 to about 35 nucleotides inlength, about 5 to about 30 nucleotides in length, about 5 to about 25nucleotides in length, about 5 to about 20 nucleotides in length, about5 to about 15 nucleotides in length, about 5 to about 10 nucleotides inlength, and all oligonucleotides intermediate in length of the sizesspecifically disclosed to the extent that the oligonucleotide is able toachieve the desired result. Accordingly, oligonucleotides of 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, and 100 nucleotides in length are contemplated.

In still other aspects, oligonucleotides comprise from about 8 to about80 nucleotides (i.e. from about 8 to about 80 linked nucleosides). Oneof ordinary skill in the art will appreciate that methods utilizecompounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 nucleotide in length.

The nanoparticles, the oligonucleotides or both are functionalized inorder to attach the oligonucleotides to the nanoparticles. Such methodsare known in the art. For instance, oligonucleotides functionalized withalkanethiols at their 3′-termini or 5′-termini readily attach to goldnanoparticles. See Whitesides, 1995, Proceedings of the Robert A. WelchFoundation 39th Conference On Chemical Research Nanophase Chemistry,Houston, Tex., pages 109-121. See also, Mucic et al., 1996, Chem.Commun. 555-557 (describes a method of attaching 3′ thiol DNA to flatgold surfaces; this method can be used to attach oligonucleotides tonanoparticles). The alkanethiol method can also be used to attacholigonucleotides to other metal, semiconductor and magnetic colloids andto the other nanoparticles listed above. Other functional groups forattaching oligonucleotides to solid surfaces include phosphorothioategroups (see, e.g., U.S. Pat. No. 5,472,881 for the binding ofoligonucleotide-phosphorothioates to gold surfaces), substitutedalkylsiloxanes (see, e.g. Burwell, 1974, Chemical Technology, 4: 370-377and Matteucci and Caruthers, 1981, J. Am. Chem. Soc., 103: 3185-3191 forbinding of oligonucleotides to silica and glass surfaces, and Grabar etal., 1995, Anal. Chem., 67: 735-743 for binding of aminoalkylsiloxanesand for similar binding of mercaptoaklylsiloxanes). Oligonucleotidesterminated with a 5′ thionucleoside or a 3′ thionucleoside may also beused for attaching oligonucleotides to solid surfaces. The followingreferences describe other methods which may be employed to attacholigonucleotides to nanoparticles: Nuzzo et al., 1987, J. Am. Chem.Soc., 109: 2358 (disulfides on gold); Allara and Nuzzo, 1985, Langmuir,1: 45 (carboxylic acids on aluminum); Allara and Tompkins, 1974, J.Colloid Interface Sci., 49: 410-421 (carboxylic acids on copper); Iler,The Chemistry Of Silica, Chapter 6, (Wiley 1979) (carboxylic acids onsilica); Timmons and Zisman, 1965, J. Phys. Chem., 69: 984-990(carboxylic acids on platinum); Soriaga and Hubbard, 1982, J. Am. Chem.Soc., 104: 3937 (aromatic ring compounds on platinum); Hubbard, 1980,Acc. Chem. Res., 13: 177 (sulfolanes, sulfoxides and otherfunctionalized solvents on platinum); Hickman et al., 1989, J. Am. Chem.Soc., 111: 7271 (isonitriles on platinum); Maoz and Sagiv, 1987,Langmuir, 3: 1045 (silanes on silica); Maoz and Sagiv, 1987, Langmuir,3: 1034 (silanes on silica); Wasserman et al., 1989, Langmuir, 5: 1074(silanes on silica); Eltekova and Eltekov, 1987, Langmuir, 3: 951(aromatic carboxylic acids, aldehydes, alcohols and methoxy groups ontitanium dioxide and silica); Lec et al., 1988, J. Phys. Chem., 92: 2597(rigid phosphates on metals). Additionally, any suitable method forattaching oligonucleotides onto the nanoparticle surface may be used. Aparticularly preferred method for attaching oligonucleotides onto asurface is based on an aging process described in U.S. application Ser.No. 09/344,667, filed Jun. 25, 1999; Ser. No. 09/603,830, filed Jun. 26,2000; Ser. No. 09/760,500, filed Jan. 12, 2001; Ser. No. 09/820,279,filed Mar. 28, 2001; Ser. No. 09/927,777, filed Aug. 10, 2001; and inInternational application nos. PCT/US97/12783, filed Jul. 21, 1997;PCT/US00/17507, filed Jun. 26, 2000; PCT/US01/01190, filed Jan. 12,2001; PCT/US01/10071, filed Mar. 28, 2001, the disclosures which areincorporated by reference in their entirety. The aging process providesnanoparticle-oligonucleotide conjugates with unexpected enhancedstability and selectivity. The method comprises providingoligonucleotides preferably having covalently bound thereto a moietycomprising a functional group which can bind to the nanoparticles. Themoieties and functional groups are those that allow for binding (i.e.,by chemisorption or covalent bonding) of the oligonucleotides tonanoparticles. For instance, oligonucleotides having an alkanethiol, analkanedisulfide or a cyclic disulfide covalently bound to their 5′ or 3′ends can be used to bind the oligonucleotides to a variety ofnanoparticles, including gold nanoparticles.

The oligonucleotides are contacted with the nanoparticles in water for atime sufficient to allow at least some of the oligonucleotides to bindto the nanoparticles by means of the functional groups. Such times canbe determined empirically. For instance, it has been found that a timeof about 12-24 hours gives good results. Other suitable conditions forbinding of the oligonucleotides can also be determined empirically. Forinstance, a concentration of about 10-20 nM nanoparticles and incubationat room temperature gives good results.

Next, at least one salt is added to the water to form a salt solution.The salt can be any suitable water-soluble salt. For instance, the saltmay be sodium chloride, magnesium chloride, potassium chloride, ammoniumchloride, sodium acetate, ammonium acetate, a combination of two or moreof these salts, or one of these salts in phosphate buffer. Preferably,the salt is added as a concentrated solution, but it could be added as asolid. The salt can be added to the water all at one time or the salt isadded gradually over time. By “gradually over time” is meant that thesalt is added in at least two portions at intervals spaced apart by aperiod of time. Suitable time intervals can be determined empirically.

The ionic strength of the salt solution must be sufficient to overcomeat least partially the electrostatic repulsion of the oligonucleotidesfrom each other and, either the electrostatic attraction of thenegatively-charged oligonucleotides for positively-chargednanoparticles, or the electrostatic repulsion of the negatively-chargedoligonucleotides from negatively-charged nanoparticles. Graduallyreducing the electrostatic attraction and repulsion by adding the saltgradually over time has been found to give the highest surface densityof oligonucleotides on the nanoparticles. Suitable ionic strengths canbe determined empirically for each salt or combination of salts. A finalconcentration of sodium chloride of from about 0.1 M to about 1.0 M inphosphate buffer, preferably with the concentration of sodium chloridebeing increased gradually over time, has been found to give goodresults.

After adding the salt, the oligonucleotides and nanoparticles areincubated in the salt solution for an additional period of timesufficient to allow sufficient additional oligonucleotides to bind tothe nanoparticles to produce the stable nanoparticle-oligonucleotideconjugates. As will be described in detail below, an increased surfacedensity of the oligonucleotides on the nanoparticles has been found tostabilize the conjugates. The time of this incubation can be determinedempirically. A total incubation time of about 24-48, preferably 40hours, has been found to give good results (this is the total time ofincubation; as noted above, the salt concentration can be increasedgradually over this total time). This second period of incubation in thesalt solution is referred to herein as the “aging” step. Other suitableconditions for this “aging” step can also be determined empirically. Forinstance, incubation at room temperature and pH 7.0 gives good results.

The conjugates produced by use of the “aging” step have been found to beconsiderably more stable than those produced without the “aging” step.As noted above, this increased stability is due to the increased densityof the oligonucleotides on the surfaces of the nanoparticles which isachieved by the “aging” step. An alternative “fast salt aging” processproduced particles with comparable DNA densities and stability. Byperforming the salt additions in the presence of a surfactant, forexample approximately 0.01% sodium dodecylsulfate (SDS), Tween, orpolyethylene glycol (PEG), the salt aging process can be performed inabout an hour.

The surface density achieved by the “aging” step will depend on the sizeand type of nanoparticles and on the length, sequence and concentrationof the oligonucleotides. A surface density adequate to make thenanoparticles stable and the conditions necessary to obtain it for adesired combination of nanoparticles and oligonucleotides can bedetermined empirically. Generally, a surface density of at least 10picomoles/cm² will be adequate to provide stablenanoparticle-oligonucleotide conjugates. Preferably, the surface densityis at least 15 picomoles/cm². Since the ability of the oligonucleotidesof the conjugates to hybridize with nucleic acid and oligonucleotidetargets can be diminished if the surface density is too great, thesurface density is preferably no greater than about 35-40 picomoles/cm².Methods are also provided wherein the oligonucleotide is bound to thenanoparticle at a surface density of at least 10 pmol/cm², at least 15pmol/cm², at least 20 pmol/cm², at least 25 pmol/cm², at least 30pmol/cm², at least 35 pmol/cm², at least 40 pmol/cm², at least 45pmol/cm², at least 50 pmol/cm², or 50 pmol/cm² or more.

“Hybridization,” which is used interchangeably with the term “complexformation” herein, means an interaction between two or three strands ofnucleic acids by hydrogen bonds in accordance with the rules ofWatson-Crick DNA complementarity, Hoogstein binding, or othersequence-specific binding known in the art. Alternatively it can mean aninteraction between polypeptides as defined herein in accordance withsequence-specific binding properties known in the art. Hybridization canbe performed under different stringency conditions known in the art.Under appropriate stringency conditions, hybridization between the twocomplementary strands or two polypeptides could reach about 60% orabove, about 70% or above, about 80% or above, about 90% or above, about95% or above, about 96% or above, about 97% or above, about 98% orabove, or about 99% or above in the reactions.

In various aspects, the methods include use of two or threeoligonucleotides or polypeptides which are 100% complementary to eachother, i.e., a perfect match, while in other aspects, the individualoligonucleotides are at least (meaning greater than or equal to) about95% complementary to each over the all or part of length of eacholigonucleotide, at least about 90%, at least about 85%, at least about80%, at least about 75%, at least about 70%, at least about 65%, atleast about 60%, at least about 55%, at least about 50%, at least about45%, at least about 40%, at least about 35%, at least about 30%, atleast about 25%, at least about 20% complementary to each other.

It is understood in the art that the sequence of the oligonucleotideused in the methods need not be 100% complementary to each other to bespecifically hybridizable. Moreover, oligonucleotide may hybridize toeach other over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). Percent complementarity between anygiven oligonucleotide can be determined routinely using BLAST programs(Basic Local Alignment Search Tools) and PowerBLAST programs known inthe art (Altschul et al., 1990, J. Mol. Biol., 215: 403-410; Zhang andMadden, 1997, Genome Res., 7: 649-656).

In one aspect, methods are provided wherein the packing density of theoligonucleotides on the surface of the nanoparticle is sufficient toresult in cooperative behavior between nanoparticles and betweenpolynucleotide strands on a single nanoparticle. In another aspect, thecooperative behavior between the nanoparticles increases the resistanceof the oligonucleotide to degradation.

As used herein, “stable” means that, for a period of at least six monthsafter the conjugates are made, a majority of the oligonucleotides remainattached to the nanoparticles and the oligonucleotides are able tohybridize with nucleic acid and oligonucleotide targets under standardconditions encountered in methods of detecting nucleic acid and methodsof nanofabrication.

Each nanoparticle utilized in the methods provided has a plurality ofoligonucleotides attached to it. As a result, eachnanoparticle-oligonucleotide conjugate has the ability to hybridize to asecond oligonucleotide that is conjugated to a fluorophore detectablydistinct from the fluorophore present on the firstnanoparticle-oligonucleotide conjugate and functionalized on a secondnanoparticle, and when present, a free oligonucleotide, having asequence sufficiently complementary. In one aspect, methods are providedwherein each nanoparticle is functionalized with identicaloligonucleotides, i.e., each oligonucleotide attached to thenanoparticle has the same length and the same sequence. In otheraspects, each nanoparticle is functionalized with two or moreoligonucleotides which are not identical, i.e., at least one of theattached oligonucleotides differ from at least one other attachedoligonucleotide in that it has a different length and/or a differentsequence.

The term “oligonucleotide” or “polynucleotide” includes those wherein asingle sequence is attached to a nanoparticle, or multiple copies of thesingle sequence are attached. For example, in various aspects, anoligonucleotide is present in multiple copies in tandem, for example,two, three, four, five, six, seven eight, nine, ten or more tandemrepeats.

Alternatively, the nanoparticle is functionalized to include at leasttwo oligonucleotides having different sequences with the proviso thateach oligonucleotide is labeled with a detectably distinct marker. Asabove, the different oligonucleotide sequences are in various aspectsarranged in tandem and/or in multiple copies. Alternatively, theoligonucleotides having different sequences are attached directly to thenanoparticle. In methods wherein oligonucleotides having differentsequences are attached to the nanoparticle, aspects of the methodsinclude those wherein the different oligonucleotide sequences hybridizeto different regions on the same polynucleotide.

The oligonucleotides on the nanoparticles may all have the same sequenceor may have different sequences that hybridize with different portionsof the polynucleotide attached to another nanoparticle. Whenoligonucleotides having different sequences are used, each nanoparticlemay have all of the different oligonucleotides attached to it or thedifferent oligonucleotides are attached to different nanoparticles.Alternatively, the oligonucleotides on each of the nanoparticles mayhave a plurality of different sequences, at least one of which musthybridize with a portion of the polynucleotide on a second nanoparticle.

Nano-Flare Technology

In an aspect of the present invention, an oligonucleotide or polypeptidecontaining the recognition sequence in the binding moiety that isattached to the nanoparticle as described herein. “Recognition sequence”as used herein is understood to mean a sequence that is partially orcompletely complementary to a target molecule of interest.

The nanoparticle with attached oligonucleotide binding moiety thatcontains a recognition sequence is initially associated with a reportersequence. As used herein, a “reporter sequence” is understood to mean asequence that is partially or completely complementary and thereforeable to hybridize to the binding moiety and its recognition sequence.The reporter sequence is labeled as discussed herein above, and is alsoreferred to as a nano-flare. Further, the reporter sequence is invarious aspects comprised of fewer, the same or more bases than therecognition sequence, such that binding of the recognition sequence inthe binding moiety to its target molecule causes release of thehybridized reporter sequence, thereby resulting in a detectable andmeasurable change in the label attached to the reporter sequence (FIG.2).

In one specific aspect, nanoparticles functionalized with a recognitionsequence for a specific target mRNA are hybridized with a shortcomplementary Cy5 labeled reporter polynucleotide having a reportersequence and where the fluorescence of the Cy5 portion is quenched whenhybridized to the recognition sequence on the nanoparticle. Thisreporter sequence is also capable of being displaced by the target mRNA.Upon displacement, the Cy5 portion is no longer quenched and fluoresces,allowing for detection and quantification of a fluorescent signal, whichis correlated to the amount of target sequence hybridized to therecognition sequence with concomitant displacement of the reportersequence.

Nano-flares take advantage of the unique optical properties of goldnanoparticles (Au NPs). Au NPs quench fluorescence with a greaterefficiency (Dubertret et al., 2001, Nat. Biotechnol. 19: 365-370) andover greater distances (Dulkeith et al., 2005, Nano Lett. 5: 585-589)than molecular quenchers. Likewise, all other types of nanoparticlesdescribed herein may be used as long as they are able to quench thedetectable marker of an attached binding moiety.

Those of skill in the art are able to determine relative meltingtemperatures and/or hybridization conditions in the case of in vitrostudies without undue experimentation that will facilitate reporterbinding to the recognition sequence in the absence of the targetmolecule while resulting in displacement of said reporter sequence inthe presence of said target molecule.

The invention is illustrated by the following examples, which are notintended to be limiting in any way.

Examples Example 1

This example is meant to demonstrate that fluorescently labeledoligonucleotide-modified gold nanoparticle agents can be used to detectintracellular molecule targets. As a proof-of-concept, it isdemonstrated that intracellular detection of mRNA targets in twocell-types using fluorescently labeled oligonucleotide-modified goldnanoparticles is highly effective. These agents readily enter the cellsand produce a fluorescent signal that can easily be read using bothfluorescent microscopy and flow-cytometry.

Specifically, 13 nm gold nanoparticles were modified with severaldifferent sequences that are terminated on one end with a thiol moiety,on the other end with a fluorescent dye, and contain astructure-switching recognition sequence. In the absence of the target,the dye molecule is in close proximity with the gold nanoparticlesurface, which leads to quenching and no fluorescent signal. In thepresence of the target, the dye molecule is separated from the goldnanoparticle surface and a fluorescent signal is observed (FIG. 1.).

Au NPs were functionalized with thiolated oligonucleotides containing an18-base recognition element to a specific RNA transcript (FIG. 1) viagold thiol bond formation (Love et al., 2005, Chem. Rev. 105:1103-1169). Oligonucleotide functionalized Au NPs were then allowed tohybridize with short cyanine (Cy5) dye-terminated reporter sequencescapable of acting as “flares” when displaced by a longer target ortarget region (FIG. 1). In the bound state, the Cy5 fluorescence of thereporter strand is quenched due to proximity to the Au NP surface. Inthe presence of a target, the flare strand is displaced and liberatedfrom the Au NP by forming the longer and more stable duplex between thetarget and the oligonucleotide-modified Au NP.

Example 2

To further exemplify the use of the gold nanoparticles' ability to entercells and detect intracellular target molecules, in vitro cell cultureexperiments were carried out.

C166 mammalian cells that stably express the enhanced green fluorescenceprotein were maintained in Dulbecco's Modified Eagles Medium with 10%serum at 37° C. and 5% CO₂ and dosed with fluorescently labeledoligonucleotide-modified gold nanoparticle agents that target theenhanced green fluorescence (EGFP) protein mRNA. SKBR3 human breastcancer (vide infra) and C166 mouse endothelial cells were obtained fromthe American Tissue Culture Collection (ATCC) and were grown in McCoy's5A Medium and Dulbecco's Modified Eagles medium (DMEM), respectively,with 10% heat inactivated fetal bovine serum and maintained at 37° C. in5% CO₂. Cells were seeded in 6 or 24 well plates and grown for 1-2 daysprior to treatment. On the day of treatment, the cells wereapproximately 50% confluent. The media was replaced with fresh mediacontaining the functionalized Au NPs.

Control experiments were performed with particles containing targetingregions for the anthrax RNA, which is not present in mammalian cells.After transfection for 16 hour, these EGFP-expressing cells treated withthe EGFP targeting probes displayed a bright fluorescent signal, muchgreater than the signal observed in the control particles. As a furthercontrol experiment, the particles were tested in C166 cells that do notexpress EGFP and hence do not contain the EGFP mRNA target. In theseexperiments neither probe was found to signal once inside the cells,thus confirming that fluorescently labeled oligonucleotide-modified goldnanoparticle agents can be used to detect specific intercellularmolecules targets.

The probe entry into the cells was confirmed using inductivity-coupledplasma mass spectrometry in order to quantify the uptake and also ruleout any sequence dependent uptake effects. These data confirm that aftera typical experiment, the cells contain approximately 100,000 goldnanoparticles, and that the C166 cells take-up a similar number of goldnanoparticles regardless of the recognition sequence contained in theoligonucleotides.

Example 3

The fluorescently labeled oligonucleotide-modified gold nanoparticleagents were further examined for their oligonucleotide loading andfluorescence signaling ability. The results of these experiments confirmthat each gold nanoparticle is functionalized with approximately 60fluorescent oligonucleotides that contain the recognition sequence.Furthermore, when a 1 nM solution of the variousoligonucleotide-modified gold nanoparticle agents were digested in a KCNsolution, they all displayed a nearly identical florescence. Takentogether this characterization data indicates that in the absence oftarget, the fluorescently labeled oligonucleotide-modified goldnanoparticle agents display an identical fluorescence signal, furtherconfirming that the intracellular signaling observed was caused by aspecific intracellular binding event.

The detection of endogenous genes is of particular importance for drugdiscovery and genetic research. Thus, gold nanoparticles were preparedthat can be used to sense the presence of the cancer gene survivin.These particles, when compared control sequences, again display a brightfluorescent signal from inside survivin expressing A549 lung cancercells. These results indicate that the described fluorescently labeledoligonucleotide-modified gold nanoparticle agents can be used todirectly read out the presence of a native mRNA target.

101071 The fluorescence signals that were observed can alternatively bedetected in large populations of the treated cells using a simple,bench-top flow cytometer instrument. These experiments again highlightthe very efficient uptake efficiency that is observed for thesefluorescently labeled oligonucleotide-modified gold nanoparticle agents,and also that nearly all cells in a given sample show strong signalindicating the presence of an intracellular mRNA target. Here, 1000 cellcounts were plotted as a function of their fluorescence intensity usinga Guava Easy Cyte flow cytometer and the instruments software. In theseexperiments it was observed that a dramatic shift in the fluorescence ofthe population of the survivin expressing A549 cells was seen when theywere treated with the survivin targeting fluorescently labeledoligonucleotide-modified gold nanoparticle agents, relative to thosetreated with the control anthrax targeting agents. The results indicatethat when coupled to flow-cytometry, fluorescently labeledoligonucleotide-modified gold nanoparticle agents are well-suited forsorting large populations of cells.

Also compared was the efficiency of the particle with a conventionalquencher-fluorophore oligonucleotide sequence that has been transfectedinto the cells with the Lipofectamine 2000 formulation. Under theanalogous conditions where our particles showed dramatic signalingability, these formulations show a negligible signaling ability. Evenwhen their concentration was increased 10 times, they showed little orno signal, thus proving that under these conditions, the nanoparticlesoutperform a conventional quencher-fluorophore oligonucleotide probe.

Taken in sum, the foregoing examples show that:

1) The gold nanoparticles assist in the intracellular delivery of afluorophore containing oligonucleotide that is capable of detecting atarget.

2) These fluorescently labeled oligonucleotide-modified goldnanoparticle agents can be used to detect both endogenous and exogenousintracellular targets.

3) The fluorescent signal that indicates the presence of the specificmRNA target can be read by either a fluorescent microscope or aflow-cytometer.

4) The efficient uptake of these agents and their high signaling abilitymakes them well suited for sorting large cell populations.

5) The efficient uptake of these agents and their high signaling abilitysurpasses conventional quencher-fluorophore oligonucleotide probe underthe conditions studied.

6) The principles can be extended to other structure-switchingrecognition sequences such as nucleic acid aptamers and peptides.

Additional fluorescently labeled aptamer-containing particle probes thattarget the molecule adenosine triphosphate (ATP) are also contemplatedby the present invention.

The present invention also contemplates the ability to simultaneouslydetect multiple intracellular targets, and quantify their intracellularconcentrations in real time. The principles can be applied to real-timemonitoring cell function in higher organisms.

Example 4

Nano-flares have been prepared using 13 nm Au NPs, since this sizeparticle is an efficient quencher, can be densely functionalized witholigonucleotides (Mirkin et al., 1996, Nature 382: 607-609), and doesnot efficiently scatter visible light, which is important for designingoptical probes with minimal interference.

Au NPs were functionalized with thiolated oligonucleotides containing arecognition element to a specific RNA transcript (FIG. 2) via gold thiolbond formation (Love et al., 2005, Chem. Rev. 105: 1103-1169).Oligonucleotides were synthesized on an Expedite 8909 NucleotideSynthesis System (ABI) using standard solid-phase phosphoramiditemethodology. Bases and reagents were purchased from Glen Research.Oligonucleotides were purified by reverse-phase high performance liquidchromatography (HPLC). To prepare nano flare probes, citrate-stabilizedgold nanoparticles (13±1 nm) were prepared using published procedures(Frens, G., 1973, Nature-Physical Science 241: 20-22. Thiol-modifiedoligonucleotides were added to 13±1 nm gold colloids at a concentrationof 3 nmol of oligonucleotide per 1 mL of 10 nM colloid and shakenovernight. After 12 hours, sodium dodecylsulfate (SDS) solution (10%)was added to the mixture to achieve a 0.1% SDS concentration. Phosphatebuffer (0.1 M; pH 7.4) was added to the mixture to achieve a 0.01 Mphosphate concentration, and six aliquots of sodium chloride solution(2.0 M) were then added to the mixture over an eight-hour period toachieve a final sodium chloride concentration of 0.15 M. The mixture wasshaken overnight to complete the functionalization process. The solutioncontaining the functionalized particles was centrifuged (13,000 rpm, 20min) and resuspended in phosphate buffered saline (PBS; 137 mM NaCl, 10mM Phosphate, 2.7 mM KCl, pH 7.4, Hyclone) three times to produce thepurified Au NPs used in all subsequent experiments. The concentration ofthe particles was determined by measuring their extinction at 524 nm(ε=2.7×10⁸ L mol⁻¹ cm⁻¹). Purified, oligonucleotide functionalized AuNPs were suspended to a concentration of 10 nM in PBS (PBS; 137 mM NaCl,10 mM Phosphate, 2.7 mM KCl, pH 7.4, Hyclone) containing 100 nM of thecomplementary Cy5 labeled reporter sequence. The mixture was heated to70° C., slowly cooled to room temperature, and stored in the dark for atleast 12 hours to allow hybridization. Particles were filter sterilizedusing a 0.2 μm acetate syringe filter (GE Healthcare). Theoligonucleotide sequences that were used are as follows:

Recognition Sequence: (SEQ ID NO. 1) 5′-CTT GAG AAA GGG CTG CCA AAAAA-SH-3′ Reporter Sequence: (SEQ ID NO. 2) 3′-CCC GAC GGT T-Cy5-5′Target Region: (SEQ ID NO. 3) 3′-GAA CTC TTT CCC GAC GGT-5′

Nano-flare probes or molecular beacons were diluted to a concentrationof 1 nM in PBS containing 0.1% Tween 20 (Sigma) and treated with acomplementary target (target concentration, 1 μM). The fluorescencespectra were recorded on a Jobin Yvon Fluorolog Fl-3-22 exciting at 633nm and measuring emission from 650 to 750 nm in 1 nm increments.Oligonucleotide functionalized Au NPs were then allowed to hybridizewith short cyanine (Cy5) dye-terminated reporter sequences capable ofacting as “flares” when displaced by a longer target or target region.In the bound state, the Cy5 fluorescence of the reporter strand isquenched due to proximity to the Au NP surface. In the presence of atarget, the flare strand is displaced and liberated from the Au NP byforming the longer and more stable duplex between the target and theoligonucleotide-modified Au NP.

Testing the nano-flare design using synthetic complementary targetsdemonstrates that the probes respond with a 3.8-fold increase influorescence signal upon target recognition and binding. In contrast,the signal does not change in the presence of a non-complementarytarget, and is of comparable magnitude to background fluorescence. Theseresults thus demonstrate that nano-flares are efficient at signaling thepresence of a specific target.

Example 5

Having established the signaling ability of nano-flare probes withsynthetic targets, their ability to enter, visualize and detect RNAtargets in live cells was investigated. Nano-flares were designed toincorporate a complementary region for the survivin transcript, a targetthat has received significant attention due to its potential use incancer therapeutics and diagnostics (Altieri et al., 2003, Oncogene 22:8581-8589). The SKBR3 cell line (human breast cancer), which expresses ahigh number of survivin transcripts (Peng et al., 2005, Cancer Res. 65:1909-1917), was used as a model to test survivin-targeting nano-flares.The survivin recognition and reporter sequences are as shown above (SEQID NO. 1 and SEQ ID NO. 2). As a control, a second probe containing anon-complementary sequence was prepared. The non-complementary probe wasdesigned and determined to have similar background fluorescence, meltingproperties, and signaling ability as the survivin probe. The survivincontrol probe oligonucleotide sequence was:

Control particle recognition sequence: (SEQ ID NO. 4) 5′-CTA TCG CGT ACAATC TGC AAA AA-SH-3′ Control particle reporter sequence: (SEQ ID NO. 5)3′-GCA TGT TAG ACG T-Cy5-5′ Survivin molecular beacon: (SEQ ID NO. 6)5′-Cy5-CGA CGG AGA AAG GGC TGC CAC GTC G dabcyl-3′ Control molecularbeacon, (SEQ ID NO. 7) 5′-Cy5-CGA CGT CGC GTA CAA TCT GCC GTCG-dabcyl-3′

Cells were cultured on glass microscope cover slips, incubated withnano-flares, and imaged using scanning confocal microscopy.Specifically, cells were grown on glass coverslips placed at the bottomof 6 well tissue culture plates. After 1 day, the media was replacedwith media containing nano-flares (particle concentration, 125 pM).After 6 hours of treatment, the media was replaced, and the cells werecultured for an additional 12 hours. The coverslips were removed, washedwith PBS, and fixed to a chamber filled with PBS mounted on a glassslide. All images were obtained with a Zeiss 510 LSM at 63×magnification using a 633 nm HeNe laser excitation source.

101181 SKBR3 cells treated with survivin nano-flares were highlyfluorescent as compared to those treated with the non-complementarycontrols. To further confirm that this signaling is consistent with thepresence of survivin, a C166 cell-line (mouse endothelial) was used as acontrol since it does not contain the human survivin transcript. C166cells were treated with both the survivin and control probes. In thiscase, no distinguishable difference in the fluorescence of the cells wasobserved after treatment. These imaging results were consistent withreverse transcriptase PCR (RT-PCR) measurements (vide infra).

In order to quantify the intracellular signaling of the nano-flares,cells treated with probes were examined using analytical flow-cytometry.Additionally, flow cytometry allows one to collect fluorescence data fora large population of cells. This eliminates variations and experimentalartifacts that can be observed using techniques such as fluorescenceimaging which only permit the examination of a small sample of cells.Cells were treated with nano-flares as described above (particleconcentration, 10 nM). Molecular beacon probes (SEQ ID NO. 6 and SEQ IDNO. 7) were delivered to cells using Lipofectamine 2000 (Invitrogen).After treatment, cells were detached from culture flasks using trypsin.Flow cytometry was performed using a DakoCytomation CyAn, exciting at635 nm.

Cell-lines transfected with nano-flares showed uniform singlepopulations of fluorescent cells, consistent with the greater than 99%cell penetration that we observe when transfecting antisense particles(Rosi et al., Science 312: 1027-1030). Flow-cytometry revealed thatSKBR3 cells treated with survivin nano-flares were highly fluorescentand 2.5 times more fluorescent than the population treated withnon-complementary controls. For comparison, in C166 cell models, bothprobes resulted in a similar low fluorescent signal. These flowcytometry experiments are in excellent agreement with confocal imagingand demonstrate the uniform cellular internalization and intracellularsignaling of the nano-flares.

Experiments then were designed to understand the unique properties ofthese probes in the context of intracellular detection experiments.First, the intracellular performance of nano-flares was compared with amolecular beacon reporter delivered using Lipofectamine, a commercialtransfection agent (Peng et al., 2005, Cancer Res. 65: 1909-1917; Nitinet al., 2004, Nucleic Acids Res. 32: e58). Molecular beacons andnano-flares were introduced to SKBR3 cells (transfection concentration,10 pM) and their signal abilities were studied using flow cytometry.Cells treated with survivin nano-flares produced 55 times greaterfluorescence signal than those treated with survivin molecular beaconprobes transfected at the same concentration. Fluorescence measurementsoutside of the cell culture indicate that each nano-flare probe containsapproximately 10 fluorophores and therefore could be expected topotentially have a 10 times greater signal than the molecular beacon atequal probe concentrations. The larger than expected intracellularfluorescence suggests that nano-flares are internalized more rapidly orto a greater extent than the molecular beacon probes.

Next, molecular beacons were transfected at high concentration (0.5 nM)to achieve an intracellular fluorescence signal to that observed withthe nano-flares. The background fluorescence contributed by thenon-complementary probes was compared (both molecular beacon andnano-flare). The fluorescence of the non-complementary molecular beaconprobe is significantly greater than that of the non-complementarynano-flare. Since the difference between the background and signal iscritical for accurate target detection, the lower background ofnano-flares provides an important advantage when detecting intracellulartargets.

To probe how enzymatic degradation leads to non-specific signaling,nano-flares were incubated with the endonuclease DNAse I (0.38 mg/L, aconcentration significantly greater than what would be found inacellular environment), and measured the rate of degradation bymonitoring the increase in fluorescence signal as a function of time.Nano-flare probes were diluted to a concentration of 2.5 nM in PBS (pH7.0), 0.25 mM MgCl₂ and 50 mg/L Bovine Serum Albumin (FischerScientific). Bovine Pancreatic DNase I (United States Biochemical) wasadded immediately before reading (concentration, 0.38 mg/L). Allexperiments were preformed on a Photal Otsuka Electronics FluoDia T70with excitation at 620 nm and emission at 665 nm. Molecular beacons weretested in an analogous manner at a concentration of 25 nM. Theapproximate rates of degradation under these experimental conditionswere determined from the slope of the linear region of the degradationcurves (Rizzo et al., 2002 Molecular and Cellular Probes 16: 277-283.

The results of the assay reveal that the nano-flare is degraded at anormalized rate of 0.275 nmol min⁻¹ under these conditions. Incomparison, a molecular beacon is degraded at a normalized rate of 1.25nmol min⁻¹, approximately 4.5 times more rapidly than the nano-flare.Since nuclease activity leads to increased background fluorescence in aconventional probe, the reduced nuclease activity of the nano-flaresleads to a system with lower background signal.

Example 6

To demonstrate an application where the cellular entry, elevatedsignaling, and low background of the nano-flare translate into a highsensitivity for changes in intracellular amounts of RNA, siRNA knockdownexperiments were conducted to reduce the levels of survivin RNAtranscripts in the SKBR3 cell models. siRNA against human survivin(Santa Cruz) was delivered to cells using Lipofectamine 2000(Invitrogen) when cells were approximately 50% confluent (siRNAconcentrations, 20, 40, and 80 nM). After 24 hours, the media waschanged with media containing the nano-flare probes (particleconcentration, 50 pM). After 6 hours, the cells were washed and freshmedia was added. Cells were cultured for an additional 12 hours andanalyzed using flow cytometry.

Cells were initially treated with siRNA against survivin, and theintracellular RNA levels were quantified using nano-flares and flowcytometry. An siRNA concentration-dependent shift in the fluorescence ofthe cell population was observed as a function of the concentration ofsiRNA added to the cell culture (FIG. 3 a). The siRNA concentration isgiven in the graph to the left of the histogram. In the untreatedsample, half of the population exhibiting equal or greater fluorescencethan the mean is shaded grey. Treated samples show a smaller fraction ofthe cell population exhibiting the mean fluorescence (declining grey,increasing black). To confirm that this shift was commensurate with adecrease in the number of survivin transcripts, RT-PCR measurements wereconducted on samples treated with the same concentrations of siRNA.Cells were counted using a Guava EasyCyte Mini (Guava Technologies).Total RNA was isolated from the cell using an RNeasy Plus Kit (Qiagen)following the manufacturers protocol. During the cell lysis step, 5×10⁷copies of Enhanced Green Fluorescent Protein (EGFP) RNA were added toeach sample to account for RNA loss during isolation and purification.To generate RNA standard curves for qRT-PCR, the fragments of RNA to bequantified were generated from the appropriate cellular RNA. Using PCRand primers containing a T7 promoter site, we converted the fragmentsinto transcription compatible sequences (DNA 4 RNA). The transcriptswere purified using the MEGAclear kit (Ambion) following themanufacturer's protocol. RNA concentration was measured using theRibogreen RNA quantification kit (Invitrogen), and a dilution series ofstock RNA was used to generate a standard curve. Primers were:

EGFP Forward (SEQ ID NO. 8) 5′-TCT TCT TCA AGG ACG ACG GCA ACT-3′ EGFPReverse (SEQ ID NO. 9) 5′-TGT GGC GGA TCT TGA AGT TCA CCT-3′ T7 EGFPForward (SEQ ID NO. 10) 5′-TGC ATA ATA CGA CTC ACT ATA GGG AGA TCT TCTTCA AGG ACG ACG GGC AAC T-3′ Survivin Forward (SEQ ID NO. 11) 5′-ATG GGTGCC CCG ACG TTG-3′ Survivin Reverse (SEQ ID NO. 12) 5′-AGA GGC CTC AATCCA TGG-3′ T7 Survivin Forward (SEQ ID NO. 13) 5′-TGC ATA ATA CGA CTCACT ATA GGG AGA TGG GTG CCC CGA CGT TG-3′

Quantitative-PCR and analysis were preformed using LightCycler RNAmaster SYBR green kits (Roche Applied Sciences) according to themanufacturer's recommendation. Reverse transcription was allowed toproceed at 61° C. for 20 minutes, followed by 45 amplification cycles(95° C., 5 sec; 54° C., 15 sec; 72° C., 20 sec), and target gene RNA wasnormalized to the standard curves generated. All reactions were done intriplicate.

The linear decrease in the fluorescence signal within the population ofcells was in agreement with the decrease in the number of survivin RNAcopies as determined by RT-PCR measurements (FIG. 3 b). Taken together,these results indicate that the nano-flares are sensitive to changes inthe number of intracellular transcripts. The RT-PCR was conducted intriplicate, and the error bars shown above are the standard deviationsof those measurements.

A new class of intracellular probe termed “nano-flares” has beendeveloped. Nano-flares are novel and potentially very useful since theyare the only probe that combines cellular transfection, enzymaticprotection, RNA detection and quantification, and mRNA visualization. Inaddition to their demonstrated use in the context of siRNA knockdownexperiments, nano-flares are contemplated to be useful in other areassuch as cell sorting, gene profiling, and real-time drug testing.Finally, given the ability of these materials to also act as generegulation agents (Rosi et al., 2006, Science 312: 1027-1030; Seferos etal., 2007, Chem Bio Chem 8: 1230-1232), these probes are contemplated toeasily adapted to simultaneously transfect, control and visualize geneexpression in real-time.

In summary, these results demonstrate:

1) Gold nanoparticles assist in the intracellular delivery of afluorophore-containing oligonucleotide that is capable of detecting atarget.

2) These fluorescently labeled oligonucleotide-modified goldnanoparticle agents can be used to detect, visualize, and quantifyintracellular targets.

3) The fluorescent signal that indicates the presence and quantity ofspecific mRNA targets can be transduced into a readable measure.

4) The efficient uptake of these agents, and their high signalingability, and low toxicity makes them well suited for distinguishing cellpopulations.

5) The efficient uptake of these agents, and their high signalingability surpasses conventional quencher-fluorophore oligonucleotideprobes under the conditions studied.

6) The principles can be extended to other structure-switchingrecognition sequences such as nucleic acid aptamers and peptides.

The present invention contemplates that use of the probes will lead tothe ability to simultaneously detect multiple intracellular targets, andquantify their intracellular concentrations in real-time. The principlesare also contemplated to be applied to real-time monitoring of cellfunction in higher organisms, and used to concurrently delivertherapeutics while simultaneously monitoring their efficacy.

1) A method of determining the intracellular concentration of a targetmolecule comprising the step of contacting the target molecule with ananoparticle under conditions that allow association of the targetmolecule with the nanoparticle, said nanoparticle comprising a bindingmoiety specific for said target molecule, said binding moiety labeledwith a marker, wherein the association of the target molecule and thenanoparticle results in detectable change in the marker, and wherein thechange in the detectable marker is proportional to the intracellularconcentration of said target molecule. 2) The method of claim 1 whereinthe binding moiety is selected from the group consisting of apolynucleotide and a polypeptide. 3) The method of claim 2 wherein thebinding moiety is DNA. 4) The method of claim 2 wherein the bindingmoiety is RNA. 5) The method of claim 1 wherein the target molecule isselected from the group consisting of a polynucleotide and apolypeptide. 6) The method of claim 2 wherein said binding moiety is apolynucleotide covalently attached to said nanoparticle and said markeris a label attached to a polynucleotide hybridized to said bindingmoiety polynucleotide, wherein association of said binding moietypolynucleotide with said target molecule releases said hybridizedpolynucleotide and said marker is detectable after release. 7) Themethod of claim 6 wherein said label attached to said polynucleotide isquenched when said polynucleotide with said label is hybridized to saidbinding moiety. 8) The method of claim 2 wherein said binding moiety isa polynucleotide comprising a marker which is a detectable label,wherein said marker is detectable only when said polynucleotide isassociated with the target molecule. 9) The method of claim 8 whereinsaid marker is quenched when said binding moiety is not associated withsaid target molecule. 10) The method of claim 2 wherein said bindingmoiety is a polypeptide and said marker is a label associated with saidpolypeptide binding moiety, and wherein association of said bindingmoiety with said target molecule releases said label and said marker isdetectable after release. 11) The method of claim 10 wherein said labelis quenched when in association with said binding moiety. 12) The methodof claim 1 wherein said nanoparticle comprises a multiplicity of bindingmoieties. 13) The method of claim 12 wherein the binding moietiesspecifically associate with one target molecule. 14) The method of claim12 wherein the binding moieties specifically associate with more thanone target molecule.